C L I N I C A L T R I A L
Correlation of HER2 status between primary tumors
and corresponding circulating tumor cells in advanced breast
cancer patients
Marta PestrinÆ Silvia Bessi Æ Francesca Galardi Æ Mara Truglia Æ Annibale BiggeriÆ Chiara Biagioni Æ Silvia Cappadona Æ
Laura BiganzoliÆ Augusto Giannini Æ Angelo Di Leo
Received: 13 February 2009 / Accepted: 26 June 2009 Ó Springer Science+Business Media, LLC. 2009
Abstract Biocharacterization of circulating tumor cells (CTCs) in the peripheral blood of advanced breast cancer (ABC) patients may represent a real-time tumor biopsy. We assessed HER2 status on CTCs from blood samples of ABC patients. CTCs were separated and stained using the Cell-Search SystemÒ. HER2 status was assessed by immu-nofluorescence and, when technically feasible, by fluores-cence in situ hybridization. Blood samples were obtained from 66 ABC patients. Forty patients had a positive CTC sample (61%) and of these, 15 (37%) had HER2 ? CTCs. We found non-concordant results in 32% of cases: 29% (8/ 28) of HER2-negative primary tumors had HER2-positive CTCs and 42% (5/12) of HER2-positive primary tumors had HER2-negative CTCs (k = 0.278). Our study suggests that a subset of patients with HER2-negative primary
tumors develops HER2-positive CTCs during disease progression.
Keywords Breast cancer HER2 status Circulating tumor cells Predictive markers Anti-HER2 therapies
Introduction
Metastatic disease, the most common cause of death in cancer patients, is a multistep process during which tumor cells disseminate from the primary tumor site and establish secondary tumors in remote sites [1]. The detection and biocharacterization of circulating tumor cells in cancer patients may provide relevant information on the progres-sion of metastatic events and may have important impli-cations for disease prognosis and treatment choices [2].
Circulating tumor cells (CTCs) can be detected from the peripheral blood of early or metastatic breast cancer patients [3–5]. A semiautomated system, the CellSearchTM system (Immunicon Corp., Huntington valley, PA, USA), has been developed for CTCs isolation using an EpCAM antibody-based immunomagnetic enrichment and an auto-mated staining methodology [6]. This assay has been shown to have high specificity and reproducibility [7]. In landmark papers, Cristofanilli et al. have used this assay for CTCs isolation from blood samples of metastatic breast cancer patients. A major finding of these studies, carried out according to the REMARK Criteria [8], was that CTCs quantification, performed before and during treatment, predicted a response to medical therapies as early as 3–4 weeks after initiation of treatment. In addition, CTCs baseline quantification was a predictor of overall survival [9,10].
Marta Pestrin and Silvia Bessi have equally contributed to this work. Presented in part at the 6th European Breast Cancer Conference, held in Berlin April 15–19, 2008.
M. Pestrin S. Bessi F. Galardi M. Truglia C. Biagioni S. Cappadona L. Biganzoli A. Giannini A. Di Leo Translational Research Unit c/o Department of Oncology, Hospital of Prato, Istituto Toscano Tumori, Piazza dell’Ospedale 2, 59100 Prato, Italy
A. Biggeri
Department of Statistics ‘‘G. Parenti’’, University of Florence and Biostatistics Unit ISPO (Institute for Cancer Prevention and Research), Viale Morgagni 59, 50134 Florence, Italy A. Di Leo (&)
‘‘Sandro Pitigliani’’ Medical Oncology Unit, Hospital of Prato, Istituto Toscano Tumori, Piazza dell’Ospedale 2,
59100 Prato, Italy
e-mail: adileo@usl4.toscana.it DOI 10.1007/s10549-009-0461-7
Current use of targeted therapies is based on the concept that metastatic cells are linear descendants of primary tumor cells with the same biologic features as the primary tumor. However, a hallmark of breast cancer is its genetic instability [11]. CTCs may express contrasting biological features to the corresponding primary tumor cells. Hence, CTCs biocharacterization may lead to identification of specific targets and subsequently direct therapy in advanced breast cancer patients.
The HER2 gene encodes for a 185-kDa tyrosine kinase glycoprotein [12]. Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of the HER2 protein, has significantly improved the clinical outcome of HER2-positive disease [13–15]. Several studies have compared HER2 status between primary tumors and matched metastatic site samples [16–19]. All of these studies have suggested an acceptable level of concordance between HER2 status of the primary and the metastatic site samples (concordance rate: 80–94%) [16–19]. Notably, in these studies, most of the metastatic site samples were drawn at the time of first relapse. In this context, it is impossible to account for selective pressure on tumor clones progression played by different lines of treatment for metastatic disease [16–19].
In an exploratory study, Meng et al. [20] reported on nine of 24 advanced breast cancer patients, whose primary tumor was HER2 negative by FISH, carrying HER2 gene amplified CTCs. Interestingly, in this study, patients had advanced disease previously treated with multiple lines of systemic therapy. These preliminary results support the hypothesis that in approximately 30% of advanced breast cancer patients, pretreated with multiple lines of systemic therapies, a shift in HER2 status might occur when a pri-mary tumor sample is compared to circulating tumor cells isolated from the same patient.
Accordingly, we decided to prospectively evaluate the level of concordance in HER2 status between primary tumor samples and their corresponding circulating tumor cells. We collected CTCs from two different series of breast cancer patients, the first with HER2-negative and the second with HER2-positive primary tumors. The results of this study are reported in the present manuscript.
Materials and methods Study population
Patients with locally advanced or metastatic breast cancer, with known HER2 status of their primary tumor, were eligible for this study. All patients were informed about the experimental design of the study and gave written informed consent. The study was previously approved by the local
ethical board. Other eligibility criteria were ECOG per-formance status 0–3, previous systemic treatment(s) for early or advanced disease were allowed, an inter-val [ 7 days between last day of systemic treatment and day of blood sample withdrawal for CTCs evaluation (http://immunicon.com/CellSearch/CellSearch HCP.aspx). A previous diagnosis of secondary malignancy was an exclusion criterion.
A twenty milliliter sample of peripheral blood was withdrawn at the time of study entry and collected in a CellsaveTMtube (Immunicon, Huntingdon Valley, PA) that contains a specific cell preservative. Under these conditions the sample was stable at room temperature for 72 h. All samples were processed within 72 h from the withdrawal time (http://immunicon.com/CellSearch/CellSearch HCP. aspx).
Ten milliliter of blood was used for CTCs enumeration and HER2 evaluation by immunofluorescence (IF). The remaining 10 ml was used for CTCs immunomagnetic enrichment. Morphological and molecular CTC character-ization was carried out on the enriched samples.
From the primary tumor samples, histological type, nodal status, estrogen receptor (ER), progesterone receptor (PgR), Ki-67, and HER2 status were retrieved from the pathology report. All of these biomarkers were evaluated by immunohistochemistry (IHC). The primary tumor was defined as HER2? if IHC 3? (PathwayÒ HER2-clone CB11- by Ventana Medical System, Inc., Tucson, Arizona– USA) or if IHC 2? with evidence of gene amplification by fluorescence in situ hybridization (FISH) (ratio HER2/CEP 17 [ 2.2) or chromogenic in situ hybridization (CISH) (HER2 copy number [ 6) [21]. Both FISH and CISH were performed according to standard procedures (PathVysion kit for FISH by Abbott Laboratories, Downers Grove, IL-USA; Spot Light Kit for CISH by Zymed Laboratories, Inc., San Francisco, CA–dUSA).
CTCs immunomagnetic isolation and HER2 immunofluorescent staining
The methodology for automated CTCs immunomagnetic isolation and for HER2 immunofluorescent staining has been described elsewhere [6–11]. Technical details of the CellSearchÒand CellSpotterÒsystems pertaining to accu-racy, precision, linearity, and reproducibility have previ-ously been reported [6,7].
Briefly, ferro fluid particles conjugated to anti-EpCAM antibodies are used for isolation of EpCAM-positive cells using a magnetic field without centrifugation. Thereafter, the supernatant, containing unbound cells, is removed and the enriched sample is processed for fluorescent staining: nucleic acids are stained with 4, 6-diamidino-2-phenylindole (DAPI); epithelial cells are stained with anti-cytokeratin
(CK)-phycoerythrin, and leukocytes are stained with an allophycocyanin-conjugated anti-CD45 antibody. At this point, the CellSearchTMHER2 Tumor Phenotyping Reagent (ImmuniconÒ) is added to identify HER2 overexpressing CTCs.
Stained cells are analyzed on Cell Track Analyzer IITM (ImmuniconÒ), a fluorescence microscope that scans the reaction cartridge. Cells with a size of at least 4 lm pre-senting the composite CK?/DAPI?/CD45-/HER-2? phenotype are classified as HER2-positive CTCs.
In this study, a case was defined as positive for CTCs when C 2 cell/7.5 ml were isolated [6]. A CTCs-positive case was defined as HER2-positive when at least 50% of CTCs were HER2 positive by IF. The arbitrary selected 50% cut-off could potentially identify true HER2-positive cases on CTCs.
Quality control was maintained via the CellSearchTM Circulating Tumor Cell Control Kit used to check reagents, instruments, and operator technique. Moreover, to test the reliability of the HER2 Tumor Phenotyping Reagent (Im-municonÒ), blood samples from healthy donors were spiked with HER2-positive tumor cells (SBRK3 cell line). Slide preparation for CTC morphological
and molecular analysis
Ten milliliter of blood was used for standard cytology and molecular analysis. The CellSearch Profile kitÒ (Immuni-conÒ) was used for automated immunomagnetic isolation of cells of interest without the staining procedures. The cell suspension obtained was subsequently cytocentrifugated and the slide was fixed in Carnoy’s fixative (3:1 methanol: glacial acetic acid) for 10 min. Papanicolaou staining was done by standard protocol, and stained cells were examined by a cytopathologist.
The PathVysion HER2/neu probe kitÒ (Abbott labora-tories) was used for the FISH analysis applied on a stained or white slide. Cases were interpreted as amplified when the ratio of HER2/CEP17 signals was greater than 2.2 [21].
Statistical analysis
Data are presented in tables of frequencies (see Tables3and
4). To assess the diagnostic performance of IF for HER2 status, we calculated sensitivity, specificity, and likelihood ratios, taking the FISH method as the gold standard. Crude percentage of correct classification and Cohen’s Kappa as chance-adjusted index of agreement are reported. Inference on Kappa values is done using standard error under the alternative hypothesis following Fleiss et al. [22].
To assess agreement on HER2 status between primary tumor and CTCs, we calculated Cohen’s Kappa and 95% confidence intervals as mentioned earlier.
Considering that HER2 status on CTCs was evaluated by IF (i.e., a nonstandardized method), we also calculated corrected Kappa values taking into account a non-differ-ential misclassification [23].
Results
CTCs count and cytopathology examination
Between June 2006 and December 2007, 66 patients with locally advanced (14 patients) or metastatic (52 patients) breast cancer were considered potentially eligible for the study. Of the metastatic patients, the vast majority had progressive disease at the time of study entry.
Among the 66 patients, 40 (61%) were CTCs positive. The median and the corrected mean of CTCs number were 5 and 85 cells/7.5 ml of blood, respectively (range: 0–60.000). Thirty-three patients (50%) had at least 5 cells/7.5 ml of blood.
Clinical and biological characteristics of the study population by CTCs count are shown in Table1.
Table2 reports main clinical characteristics of meta-static breast cancer patients by CTCs count. No clinically relevant correlations were found.
Twenty-nine CTCs-positive cases were characterized morphologically by Papanicolaou staining. It was observed that CTCs differ from cells of the corresponding primary tumor tissue. In our study, two cell types were observed: the most common was characterized by small, rounded cells with a high nucleus/cytoplasmic ratio, either isolated (sim-ilar to blood cells) or arranged in clusters; the less common was characterized by larger and sometimes elongated cells (Fig.1a–b).
HER2 protein expression and gene amplification on CTCs
Among the 40 CTCs-positive patients, 25 (63%) were classified as HER2 negative and 15 (37%) as HER2 posi-tive by IF analysis (Fig.1c–d). The mean percentage of immunofluorescence stained cells was 3 (range: 0–27%) in the HER2-negative group and 92.5 (range: 50–100%) in the HER2-positive group.
Thirty-four CTCs-positive cases were evaluated for HER2 gene amplification by FISH. Of these, nine were non-inter-pretable due to technical issues, five were HER2–amplified, and twenty were HER2-non-amplified (Fig.1e–f).
Table3summarizes the concordance of HER2 status on CTCs between IF and FISH. Taking FISH as the gold standard to define the HER2 status, IF had a sensitivity and a specificity of 80 and 95%, respectively, and a likelihood ratio of a positive and negative test equal to 16 and 0.21, respectively.
Considering that CTCs were defined as HER2 positive by IF if C 50% of cells had HER2 staining, we found that the two techniques had a 92% agreement rate, k = 0.75, P = 0.033 (95% CI: 0.422–1.000).
HER2 protein expression on CTCs and corresponding primary tumors
Table4 summarizes the results of the HER2 status com-parison between CTCs, evaluated by IF, and corresponding primary tumor, evaluated by IHC and in situ hybridization. In our series, we found 13 discordant cases (32%). Among the HER2-positive primary tumors, 42% (5/12) developed HER2-negative CTCs. These five patients had previously been treated with several lines of therapy and three of them had received trastuzumab before CTCs analysis. In three of the five HER2-negative cases on CTCs, FISH evaluation on CTCs was technically feasible confirming the HER2-negative status.
Among the HER2-negative primary tumors, 29% (8/28) developed HER2-positive CTCs. In two of the eight cases, FISH on CTCs was feasible and confirmed the HER2 positivity.
In our series, a weak concordance was found between HER2 status evaluated on primary tumor and on corre-sponding CTCs, k = 0,278 (95% CI: -0.028 to 0.584).
As HER2 status on CTCs was defined by IF (i.e., a nonstandardized method), we applied a correction for non-differential misclassification of HER2 status on CTCs using the IF sensitivity and specificity from Table3. According to this analysis, corrected kappa, using integer values, ranges from 0.309 to 0.375; thus, confirming a weak concordance.
Table5 reports main patient characteristics for the 13 cases with HER2 discordant results.
HER2 by FISH was reassessed on the primary tumor for all discordant cases, and in all, the originally assigned HER2 status was confirmed.
Discussion
In clinical practice, we tend to assume that the expression of biological markers of metastatic tumors mirrors the primary tumor biological profile.
However, tumors are biologically and clinically heter-ogeneous, and systemic therapies may play a role in selective tumor clones progression, explaining the preva-lence of a given tumor clone over the others [24].
In the past, studies have explored the concept of biolog-ical heterogeneity between the primary tumor and its met-astatic sites by comparing the expression of a given marker, most frequently HER2 or hormone receptors [16–19].
All of the reported studies had a retrospective design and, more importantly, in the vast majority of cases expression of biomarkers was evaluated on a metastatic site biopsied at the time of the first disease relapse. Of note, patients experiencing first relapse had not yet received Table 1 Patient and primary tumor characteristics by CTC count
Patients’ characteristics by CTC count CTC C 2 CTC \ 2 Overall Age (years) C50 32 (80%) 16 (62%) 48 \50 8 (20%) 10 (38%) 18 Histology Ductal 31 (71%) 19 (73%) 50 Lobular 6 (15%) 5 (19%) 11 Other 3 (8%) 2 (8%) 5 Nodal status N? 19 (47%) 18 (69%) 37 N- 11 (28%) 3 (12%) 14 Nx 10 (25%) 5 (19%) 15 ER status Pos 26 (65%) 22 (85%) 48 Neg 13 (33%) 4 (15%) 17 NA 1 (2%) 0 1 PgR status Pos 16 (40%) 17 (65%) 33 Neg 23 (58%) 9 (35%) 32 NA 1 (2%) 0 1 HER2 Pos 12 (30%) 5 (19%) 17 Neg 28 (70%) 21 (81%) 49 Overall 40 26 66
CTC circulating tumor cells, NA not available
Table 2 Main clinical characteristics of MBC patients by CTC count
CTC C 2 CTC \ 2 Total patients (N = 52) Number of metastatic sites
C3 19 (53%) 7 (44%) 26
\3 17 (47%) 9 (56%) 26
Type of metastatic sites (visceral/non-visceral)
± 3 (8%) 0 3 ; 9 (25%) 7 (44%) 16 ?/? 24 (67%) 9 (56%) 33 DFI [12 months 21 (58%) 13 (81%) 34 B12 months 15 (42%) 3 (19%) 18 ?, Presence; -, absence
MBC metastatic breast cancer, DFI disease free interval calculated from date of breast cancer diagnosis to date of first relapse
treatment for metastatic disease. In this circumstance, the potential selective pressure played by systemic therapies on tumor clone progression cannot occur. In addition, in these studies, biomarkers expression was most frequently eval-uated on a single metastatic site, assuming that this site would be representative of the bulk of metastatic disease.
The use of modern technologies allowing for the isola-tion of CTCs from the peripheral blood of cancer patients
has enabled the undertaking of studies aiming to biologi-cally characterize metastatic tumors [25–33]. Theoreti-cally, CTCs isolation and biocharacterization may become a real time and minimally invasive biopsy of the metastatic tumor and could provide the clinician with relevant infor-mation in terms of prognosis and prediction of treatment activity.
Fig. 1 Morphological analysis: panel a rounded cells with a high nucleus/cytoplasmic ratio, arranged in clusters; panel b elongated cells. Immunofluorescence analysis: panels c and d HER2, CK-PE, and DAPI windows reproduce a computer-elaborated image of the membrane, cytoplasm, and nucleus, respectively. Panel c positive
circulating tumor cell (DAPI?/CK-PE?/CD45APC-/HER2?); panel d HER2-negative circulating tumor cell (DAPI?/CK-PE?/ CD45APC-/HER2-). FISH analysis; panel e HER2-amplified circulating tumor cells; panel f HER2-non-amplified circulating tumor cells
Table 3 Concordance of HER2 status between IF and FISH eval-uated on CTC
IF positive IF negative Total
FISH positive 4 1 5
FISH negative 1 19 20
Total 5 20 25
CTC circulating tumor cells, IF immunofluorescence, FISH fluores-cence in situ hybridization
Kappa (95% CI): 0.750 (0.422–1.000)
Table 4 HER2 status comparison between primary tumors and corresponding CTC
Primary tumor CTC
HER2? by IF HER2- by IF Total
HER2? 7 5 12
HER2- 8 20 28
Total 15 25 40
CTC circulating tumor cells, IF immunofluorescence Kappa (95% CI): 0.278 (-0.028 to 0.584)
In the present study, we have used an immunomagnetic-based, semiautomated technology namely CellSearchÒ. In a clinical setting, CTCs counting by CellSearchÒhas been shown to have a prognostic value for advanced breast cancer patients [9,10]. This suggests that CTCs isolated by this technology are biologically relevant.
In this study, CTCs were isolated in only 61% of the study population. In addition, only half of the 66 locally advanced or metastatic cases reported in the present man-uscript had at least 5 cells/7.5 ml of peripheral blood. The limited number of CTCs isolated by CellSearchÒmay be poor representative of the tumor bulk of any single patient, and it may generate technical difficulties in evaluating biomarkers.
Conversely, a technology based on microchips and interaction between CTCs and anti-EpCAM-coated mi-croposts has shown to be highly sensitive in detecting relevant numbers of CTCs from peripheral blood samples of up to 99% of advanced solid tumor patients, although the clinical significance of isolated CTCs is still unclear [33].
In the present study, we have focused our efforts on the comparison of HER2 status between primary tumor and corresponding CTCs isolated from advanced breast cancer patients. HER2 is recognized as a clinically important treatment target [13–15]. The demonstration that some HER2-negative primary tumors might develop, during the course of disease progression, HER2-positive CTCs could clinically be relevant.
Our results seem to support the concept of HER2 status shifting between the primary tumor and its corresponding CTCs. The presented results are in line with previously reported data (Table6) [34–39]. Nevertheless, it is important to emphasize that the present study had as pri-mary end point the evaluation of the HER2 status shift between primary tumors and CTCs in view of a clinical trial testing anti-HER2 treatments in patients with HER2-negative primary tumors and HER2-positive CTCs. Shift-ing seems to occur in both directions. In five of twelve HER2-positive primary tumors, CTCs were defined as HER2-negative. Interestingly, in these five discordant cases the HER2/CEP17 copy numbers ratio evaluated on the primary tumor by FISH was close to the cut-off. In addi-tion, in the eight cases with HER2-negative primary tumor and HER2-positive CTCs, the number of isolated CTCs was \10/7.5 ml of blood in all but one cases.
These considerations suggest that the present results have to be taken with caution.
A further caveat of the present study is the fact that HER2 status was evaluated by IF on CTCs and by standard IHC and in situ hybridization on primary tumors. Never-theless, it is important to emphasize that in 23 of 25 cases in which both IF and FISH were technically feasible on peripheral blood samples drawn at the same time from the same patient, a concordance between the two techniques in defining the HER2 status on CTCs was found (Table3). This suggests that evaluation of HER2 by IF on CTCs may be accurate enough.
Table 5 Main patient characteristics of the discordant cases
Patient number HER2 assessment on primary tumor D time HER2 assessment by IF on CTC Previous treatmentsa
Method Result (score) Months #CTC % HER2 ? CTC CT HT
CTC011 FISH NA (0.95) 0 3 67 0 0 CTC012 FISH NA (1.36) 36 5 100 3 2 CTC018 FISH NA (1.17) 52 6 67 1 2 CTC022 FISH NA (1.02) 1 5 100 0 0 CTC023 FISH NA (1.19) 68 2 100 0 2 CTC046 FISH NA (1.01) 45 5 100 1 1 CTC048 FISH NA (0.93) 3 5 100 0 1 CTC064 FISH NA (1.07) 1 21 100 0 0 CTC010m FISH A (2.30) 12 11 27 2 1 CTC049 FISH A (2.34) 21 101 2 1 1 CTC055 FISH A (2.58) 52 18 0 0 1 CTC078 FISH A ([2.2) 96 12 8 4 2 CTC079 FISH A ([2.2) 78 37 3 1 3
CTC circulating tumor cells, # number, CT chemotherapy, HT hormonotherapy, NA non-amplified, A amplified, IF immunofluorescence, FISH fluorescence in situ hybridization
D time: time interval between date of metastatic disease diagnosis and date of CTC evaluation a For advanced disease
The results of this study generate a new question: is the evaluation of HER2 status on CTCs representative of the metastatic tumor HER2 status? This critical issue must be addressed because of its clinical relevance. Only a properly designed clinical trial can address this question. Meng et al. [20] reported that three of four heavily pretreated advanced breast cancer patients with HER2-negative primary tumors and HER2-positive CTCs had an objective tumor response to trastuzumab combined with chemotherapy. This report, although hypothesis-generating, is not strong enough to promote this strategy in the clinical practice. It was not a prospectively designed clinical trial and patients received trastuzumab in combination with chemotherapy.
Based on the results of the presently reported study, our group has recently initiated a prospectively designed phase II clinical trial, whose primary aim is the evaluation of single-agent lapatinib in patients with HER2-negative pri-mary tumors by standard criteria and HER2-positive CTCs. Whenever feasible, a biopsy from a metastatic site will be collected in parallel with CTCs biocharacterization. This will allow for correlation in HER2 status between a met-astatic tumor sample and CTCs from the same patient. We believe that the results of this ongoing trial might prag-matically address the question raised by the present study in relation to the clinical significance of HER2 status determination on CTCs.
In conclusion, the present study shows that in 32% of patients with advanced breast cancer a shift in HER2 status between the primary tumor and corresponding CTCs occurred. The shift was seen to be bidirectional. The next step is the clinical evaluation of anti-HER2 therapies in patients with negative primary tumors and HER2-positive CTCs. An ongoing Phase II trial is addressing this clinically relevant question.
Acknowledgments The authors wish to thank Ms. Elizabeth Ann Florea for the linguistic revision of the present manuscript, the As-sociazione Italiana Ricerca Cancro (AIRC)—Milan Italy, the Asso-ciazione ‘‘Sandro Pitigliani’’—Prato Italy, and the Breast Cancer Research Foundation, New York, USA, for the financial support provided to the institutional research program on CTCs in breast cancer.
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Table 6 Previous studies correlating HER2 status between primary tumor and corresponding CTC
Author Disease
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Method of HER2 evaluation Discordance rate (no) (%)
Comments Primary CTC
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Wulfing et al. [39] EBC 27 IHC on TMA ICC 48 (13/27) % of staining in HER2-positive CTC was not reported
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